Pathogenic Genes Identification Of Multiple Morphological Anomalies Of The Sperm Flagella In Human

BackgroundSurveys suggested that 8%of reproductive male population has impaired fertility.It is estimated that globally there are at least 20 million male infertility patients.The causes of male infertility contain many aspects,among which genetic factor,including single-gene disease and chromosomal aberration,accounts for over 15%of patients.Sperm activity is of vital importance to male fertility.According to the WHO data,81%of impaired male fertility is related to sperm activity,in which 20%has a directly relation with attenuated sperm activity.Severely impaired sperm activity is clinically defined as severe asthenozoospermia,which is a common cause of absolute infertility.Therefore,in-depth research of severe asthenozoospermia has both clinical value and practical significance.Currently,it is a research hot spot in the field of reproductive medicine to investigate the molecular basis of asthenozoospermia-associated male infertility from a genetic point of view,which was based on the morphology and?ltrastructure of sperm flagella.Severe asthenozoospermia can be secondary to acquired factors,including reproductive tract infections,oxidative stress,low-temperature preservation and necrospermia caused by exposure to chemicals.There is also primary severe asthenozoospermia due to patient's genetic factors.Generally,clinical doctors speculate the cause of impaired speram activity from the above two aspects.Genetic factors are speculated to be the main cause of severe asthenozoospermia involving multiple morphological abnormalities of the flagella?MMAF?,and of total immotile sperm.Up to now,there has been no report about natural conception of the spouse of an MMAP patient,neither has there been a successful case of conventional in vitro fertilization and embryo transfer?IVF-ET?pregnancy.Intracytoplasmic sperm injection?ICSI?is the only applicable assisted fertility method for MMAF patient.However,the patient's genetic defect can pass on to the next generation.Currently,the virulence gene for MMAF still remains unclear.Therefore,there is still no theoretical basis for providing genetic diagnosis and counselling for MMAF patients.Gladly,since the formal denomination of MMAF in 2014,relevant researches have been continually carried out and certain achievements were obtained.The clinical features of MMAF include:? primary infertility;? inactive or immotile sperm;? viability apparently higher than motility;? morphological evaluation by optical microscopy shows mainly flagella deformity.Multiple types of deformity can exist in the same spermatozoa,or there is similarity among the deformed flagella of different spermatozoa;? transmission electron microscopy also reveals abnormal flagella,mainly presented as absence of central microtubule and dynein arms and disorganized fibrous sheath;? ICSI is the only applicable assisted fertility method for MMAF patient and usually with good outcomes.The French researcher Ben Khelifa M and colleagues first proposed the name of MMAF in 2014 and reported 20 north-African MMAF patients,among whom 7 were found to be correlated with pathological variation of DNAH1 gene.However,in the other 13 MMAF patients,pathological variation of DNAH1 was not detected,which suggested the genetic heterogeneity of MMAF.In 2015,the Chinese scholar Shenmin Yang et al.studied the group genetic background of Han Chinese MMAF patients,who unfortunately failed to identify any potential virulence gene among the enrolled MMAF patients.This indicated the complexity of the genetic origin of MMAF,which still awaits further in-depth study.For the study of pathogenic genes of monogenic disorder,pedigree linkage analysis and positional cloning are considered to be most accurate.However,if the number of patients within the pedigree is limited or if patients are non-penetrant,incomplete penetrant or sporadic cases?comparing to heritable somatopathy,heritable infertility presents mostly as sporadic cases,very rarely showing familial aggregation?,then linkages analysis becomes restricted.The next-generation sequencing technology?NGS?is also called massively parallel signature sequencing?MPSS?or deep sequencing,which provides a novel method for studying monogenic diseases.It has a unique advantage in identifying the pathogeny of rare genetic disorders in that it neither require samples from large pedigree,nor it is biased for whole-exome and whole-genome.This study utilized NGS to identify pathogenic genetic variation of MMAF,and combined molecular biology and molecular genetics to verify the pathogenicity of the variant.ObjectiveUsing male infertility patients caused by MMAF-related severe asthenozoospermia as object of study,to screen and identify unknown variations of known MMAF-associated genes,or unknown virulence genes that might lead to MMAF,in order to provide a theoretical basis for performing clinical genetic diagnosis and counselling for MMAF patients.MethodsPart ?:We recruited 10 infertile Chinese male with MMAF.First,we focused on patient No.l?P1?who was born of consanguineous marriage.His was subject to whole-genome resequencing,followed by bioinformatics analysis based on the pattern of monogenic disorder.Homozygous variants with pathogenic potential received more attentions and known MMAF-associated genes were primarily analyzed.Secondly,the accuracy of NGS was verified by Sanger sequencing.Co-separation of phenotype and genotype was examined within his family.Next,the pathogenicity of identified variation was further studied on mRNA and protein levels using RT-PCR,Western blot and immunofluorescence confocal microscopy.Pare ?:This part focused on the 6 MMAF patients in whom no virulence genes were identified in the first part of study.First,whole exome sequencing?WES?was performed.In the annotated data,special attention was paid to genes specifically expressed in testis,and rare pathological variations were identified which were possibly related to MMAF.Secondly,Sanger sequencing confirmed the WES result,and co-segregation analysis was performed in their families.Among the 6 patients,1 patient?P10?was born of consanguineous parents.Sanger sequencing was performed on all key members of P10 family.Based on a positive sequencing result,we further performed co-separation of phenotype and genotype analysis on the common and potential MMAF-associated genes within the family.In the end,the pathogenicity of identified variations was further studied on mRNA and protein levels.ResultsPart ?:A homozygous frame-shift variation exon73:c.11726₁1727delCT was identified in the DNAH1 gene of P1.His parents and brother were all heterozygous carrier of this variation.There was phenotype and genotype co-segregation within the family.P2,P3 and P4 were also homozygous carrier of this variation,and their parents were all heterozygous carrier of this variation,which suggested there was phenotype and genotype co-segregation with the 4 patients from 3 families.The incidence of this variation in the MMAF population is 40%?4/10?.Meanwhile,western blot and immunofluorescence microscopy identified that DNAH1 protein was not expressed in the spermatozoa of MMAF patients,while expressed in spermatozoa of normal control group.Part ?:Ahomozygous splicing variation Chr10:105907757 CT/C?c.3661-2A>-?was identified in the CFAP43 gene of P10.P5 and P6 carried compound heterozygous variations in CFAP43,which were the missense variant heterozygote c.386C>A?p.Ser129Tyr?and the nonsense variant heterozygote c.2802T>A?p.Cys934*?in P5,and the heterozygous missing fragment c.3945₄431del?p.Ile1316Leufs*10?and the missense variant heterozygotec.253C>T?p.Arg85Trp?in P6.All these variants were inherited from their parents,and phenotype and genotype co-segregation was identified in the 3 families.RT-PCR and immunofluorescence microscopy experiments found that the expression of CFAP43 mRNA and protein was significantly lower in the spermatozoa of MMAF patients than in that of normal people.ConclusionPart ?:This part of study identified a novel homozygous frame-shift variation DNAH1:exon73:c.11726-11727delCT that could cause MMAF.This result indicated the complexity of the genetic pathogenesis of MMAF as well as the important role of DNAH1 in sperm motility.This is the first report of the genetic origin of MMAF-related male infertility in Han Chinese population.Our result also confirmed that the pathogenesis of MMAF has an autosomal recessive inheritance character.Part ?:This part of study suggested that pathological homozygous or compound heterozygous variations of CFAP43 could lead to MMAF,attenuate sperm motility and lead to male infertility associated with severe asthenozoospermia.In the meantime,it is also suggested that pathological variations of CFAP43 has an autosomal recessive inheritance character.In addition,our study further confirmed the genetic heterogeneity of MMAF.Novelty1.This study first showed that the pathogenesis of MMAF has an autosomal recessive inheritance character.MMAF was also shown to be an important contributor to male infertility associated with heritable severe asthenozoospermia.2.Identified a novel exon73:c.11726₁1727delCT frame-shift variation in DNAH1 that could cause MMAF,which complemented the gene mutation spectrum of DNAH1.This variation could possibly be a high-frequency variation among the Han Chinese MMAF population.We first demonstrated that pathogenic homozygote of DNAH1 variation could be inherited from parents.Heterozygotes were non-pathogenic and homozygotes were pathogenic.3.Fist identified that CFAP43 variation was associated with MMAF,and demonstrated that heterozygotes of pathogenic CFAP43 variation were non-pathogenic and homozygotes were pathogenic.4.First studied and found that within the MMAF patient population,70%of cases contain selective DNAH1 and CFAP43 biallelic loss-of-function variation,which suggested that variation of these two genes could be the main cause of MMAF.SignificanceThis study has achieved expected outcome.We first demonstrated that pathogenesis of MMAF has an autosomal recessive inheritance character.A novel frame-shift variation in DNAH1 and new biallelic variations in CFAP43 were found related to MMAP.These results could provide evidence for the development of clinical genetic diagnosis kit for MMAF,for genetic consultation and preimplantation genetic diagnosis?PGD?for MMAF patients,and for preventing the birth of MMAF babies.This studies result also certified the feasibility of combining NGS with monogenic disorder analysis in identifying the pathogenic genes of male infertility.In infertility population sharing similar sperm morphology and ?ltrastructure anomalies,annotated NGS data was combined with the high frequency feature of autosomal recessive genetic disorder in the offspring of consanguineous parents,in order to explore rare homozygous variants among testis-specific genes.These research strategies gave a valuable reference for future research of clinical doctors.Specific genetic diagnosis and precision medical intervention are the dominant mode of disease prevention and treatment.Although PGD?including PGS?assisted reproduction techniques are quickly developing in preventing the birth of babies with somatogenetic monogenic disorders and chromosomal aberrations,the etiological diagnosis,genetic consultation and precision intervention of Infertility associated with genetic factors have little progression.The result of this study hopes to promote the application of precision medicine in the field of male reproduction.